The mission of the Clinical and Molecular Immunology Group within the Clinical Immunology Section is to perform cellular, molecular and clinical studies aimed at understanding the basis of immune tolerance. A long-term objective is to develop new therapies for allograft transplantation and the treatment of autoimmune and inflammatory diseases. I. CD28 Signaling. Signaling through the CD28 receptor during T cell activation exerts a profound influence on the outcome of T cell receptor (TCR) engagement. Failure to receive a costimulatory signal through CD28 results in an unresponsive state termed anergy or in T cell death; both of which contribute to the induction and maintenance of immune tolerance. The CD28 signaling pathway is poorly understood. To decipher this pathway, we've utilized a mouse model system in which CD28 signaling is responsible for greater than 99% of T cell IL-2 production. Earlier work has shown that this CD28 dependent upregulation of IL-2 is a consequence of increased IL-2 mRNA stability. We?ve demonstrated that sequences within the 3' untranslated region of the mouse IL-2 mRNA are responsible for mRNA instability but cannot confer CD28 responsiveness upon a heterologous reporter mRNA. Additional mRNA stability elements located in exon 2 and the coding region of exon 4 are required for CD28 mediated IL-2 mRNA stabilization. Reporter constructs designed to test the role of these sequences in CD28- mediated stabilization unexpectedly revealed that IL-2 mRNA stability may be coupled to splicing of the pre-mRNA in the nucleus. While this finding has complicated our studies on the role of IL-2 exonic sequences, we plan to pursue this novel finding to determine how and why these two processes are coupled. Biochemical studies carried out to identify proteins that bind the IL-2 mRNA have focused on the 3'UTR of the mRNA. We identified one such protein to be HuR, the mammalian homolog of the Drosophila ELAV (embryonic lethal abnormal vision) gene. It is widely held that HuR binding stabilizes labile mRNAs such as c-myc and IL-3, however, we concluded that HuR binding to the IL-2 mRNA is not sufficient for CD28-mediated stabilization. This appeared in press this past year in the Journal of Biological Chemistry. The controversial nature of this observation prompted us to generate IL-2 deletion mutants expected not to bind HuR. Characterization of this mutant is incomplete, but it exhibits a partial defect in CD28-mediated stabilization indicating that HuR, if not sufficient, may be necessary for IL-2 mRNA stabilization. Bioinformatic analysis of the HuR binding site in the IL-2 mRNA reveals several potential stem-loop structures in the region and we?ve identified a second, unknown 50kD protein that binds the same sequence. In addition we?ve identified HuR binding sites downstream of the one we originally characterized. Binding to these downstream sites would by necessity be associated with a long form of the IL-2 mRNA that arises by differential polyadenylation. We are currently assessing the potential role of these additional HuR binding sites and working to identify and determine the function of the 50kD IL-2 mRNA binding protein(s). II. Molecular Consequences of IL-2 Receptor Blockade. Laboratory investigations to understand the mechanism(s) by which blockade of CD25, the high affinity IL-2 receptor (a therapeutic modality in transplantation, allergic, and autoimmune disease) inhibits immune activation have nearly completed the 2nd & 3rd stages of study. Our previous results demonstrated for the first time that both IFN-gamma production and CD40L expression are biphasic and that the latter, but not the initial phase of expression, is highly dependent of IL-2R signaling. These findings have important implications for the choice of immunosuppressive regimen (e.g. anti- IL-2R vs. anti- IL-12) employed in the setting of transplantation or autoimmune disease. These observations are being extended to understand at the molecular level the activation pathways involved in CD40L expression. Others have shown that blocking CD40L alone can induce long-term tolerance in a primate transplant model. As this has never been accomplished by blocking any other molecule, it will be of critical importance to understand the pathways controlling the expression of this important determinant of immune tolerance. We have now defined the cytokine requirements for late CD40L expression by demonstrating that IL-2 acts directly on CD40L rather than through IL-2 dependent cytokines such as IL-4 and IFN-gamma. We have also shown, that while CD40L expression on resting cells is restricted to the CD4 memory population, unexpectedly, on activated cells early and late expression occurs proportionately on naive and memory T cells. In the 3rd stage of the project, we?ve demonstrated that early CD40L expression, like late expression, is dependent on APC-T cell contact, but through different and unknown cell surface receptors. In contrast to late CD40L expression, early expression is completely independent of cytokines. The cell contact-dependent CD40L costimulatory activity of APC is present on resting cells and is actively down regulated upon APC activation, possibly through an NF-kappa B dependent pathway. The 2nd stage results have been submitted for publication and are under review. The 3rd stage results are being prepared for submission. Our current efforts are focused on identifying the unknown surface ligand on APC that is augments the induction of early CD40L expression. III. Induction of Clinical Immunotolerance in Uveitis. To date, autoimmune disease and transplant graft rejection has been managed using a barrage of immunosuppressive drugs. These medications often require life-long administration and have a plethora of serious side effects. With few exceptions, these same drugs block the induction of immune tolerance; a likely prerequisite for long term graft acceptance in the absence of continued immunosuppression, or for an autoimmune disease cure. We are currently in the middle of one trial to induce immune tolerance in uveitis and a second trial will begin later this fall pending FDA and IRB approval. Both are Phase I/II Clinical Studies to Evaluate the Induction of Immune Tolerance in Patients with Sight Threatening Autoimmune Uveitis. Study 04-EI-0115 has now been underway for a year. In this study we utilize two pharmacological agents; daclizumab, a monoclonal antibody against the interluekin-2 receptor that can control autoimmune uveitis but does not appear to block the induction of tolerance, and sirolimus, a drug that can induce experimental immune tolerance. Sirolimus is a macrocyclic lactone that inhibits T lymphocyte activation and proliferation in response to both antigenic and cytokine stimulation by a mechanism that is distinct from that of other immuno-suppressants. In cells, sirolimus binds to the immunophilin, FKBP-12, to generate an immunosuppressive complex. This complex binds to and inhibits the activation of mTOR, a key regulatory kinase. This inhibition suppresses cytokine-driven T-cell proliferation, inhibiting the progression from the G1 to S phase of the cell cycle, which is believed to responsible for tolerance induction. The study is 100% enrolled and has now reached the watershed point at which daclizumab will be tapered and discontinued. If participants can successfully be taken off daclizumab, they will remain on sirolimus alone for another 6 months before it is determined whether they have been cured of their uveitis.